Enhancing Diabetic Wound Healing Through Local Silencing of PHD2 and Activation of the AMPK Pathway

Recent data from the International Diabetes Federation (IDF) indicates that in 2017, approximately 425 million people globally were diagnosed with diabetes, with China accounting for 114 million of these cases, the highest number worldwide. One of the main complications in diabetes patients is microvascular and macrovascular lesions, which are significant risk factors for mortality and contribute to impaired wound healing. The high glucose and inflammatory environment in diabetic wounds reduces the number and function of endothelial progenitor cells, leading to a decrease in vascular endothelial growth factor (VEGF) expression and its receptors, thereby inhibiting new blood vessel formation. Additionally, vascular rupture and inflammation increase tissue oxygen consumption, exacerbating hypoxia at the wound site. Therefore, addressing hypoxia is critical for effective wound healing in diabetic patients.

Prolyl hydroxylase domain protein 2 (PHD2) is a well-known oxygen sensor and central regulator of cellular oxygen homeostasis. In the presence of oxygen, PHD2 hydroxylates hypoxia-inducible factor 1-alpha (HIF-1α), marking it for degradation. However, under hypoxic conditions, PHD2 activity decreases, stabilizing HIF-1α and activating transcription of its target genes, which promotes adaptive responses to hypoxia. Beyond its role as an HIF regulator, PHD2 also engages in other protective anti-hypoxia responses that do not depend on HIF. Activation of the PHD2 signaling pathway modulates various cellular responses to hypoxia and preconditioning stimuli, playing a crucial role in maintaining intracellular ATP levels when mitochondrial oxidative metabolism is restricted. AMP-activated protein kinase (AMPK), a key regulator of metabolic homeostasis, is activated under conditions of elevated AMP/ATP ratios, such as glucose deprivation, muscle contraction, and hypoxia. It maintains cellular energy homeostasis and adaptive responses by phosphorylating downstream proteins or influencing gene expression, thereby reducing ATP consumption and enhancing cellular tolerance to hypoxia. Based on the role of PHD2 in regulating hypoxic responses, we hypothesize that local silencing of PHD2 can promote wound healing in diabetic rats by activating the AMPK pathway.

Given that diabetic wounds are significantly hypoxic and exhibit impaired neovascularization, PHD2 was identified as a potential target for enhancing wound healing through its regulatory effects on AMPK. PHD2 is the most abundant and critical hydroxylase in most cell lines, acting as an oxygen-sensitive enzyme that links oxygen availability with cellular adaptation to hypoxia. Previous findings suggest that PHD2 can activate calmodulin-dependent protein kinase β upstream of AMPK, which is crucial for cell survival under hypoxic conditions. To test our hypothesis, we employed a lentivirus vector coated with PHD2-shRNA, provided by PackGene, to achieve local silencing of PHD2 expression in the wound tissues of diabetic model rats. This approach led to a significant improvement in wound repair. Angiogenesis was quantified by evaluating CD31-labeled vascular endothelial cells, revealing that PHD2 silencing stimulated angiogenesis within the wound. Quantitative analysis of wound tissue proteins confirmed a marked reduction in PHD2 expression in the treatment group, along with increased levels of growth factors VEGF and fibroblast growth factor-2 (FGF-2), both critical for angiogenesis. These findings indicate that PHD2 silencing enhances angiogenesis through mechanisms beyond the direct effects of VEGF or FGF-2 alone.

To explore whether AMPK activation was involved in the observed wound healing response, we conducted further experiments using two cell lines closely related to wound repair: fibroblasts, which are often dysfunctional in diabetic patients, and endothelial cells, which are integral to angiogenesis. RT-PCR and western blotting confirmed the effectiveness of PHD2 silencing via lentiviral delivery. Silencing PHD2 led to a significant increase in AMPK phosphorylation, an effect that was fully reversed by dorsomorphin, an AMPK inhibitor, without affecting PHD2 expression. This evidence supports the role of AMPK in mediating the enhanced proliferation and migration observed in both cell types when PHD2 was silenced. These effects were entirely abolished upon dorsomorphin treatment, further emphasizing the involvement of AMPK.

AMPK is a central regulator of metabolic homeostasis and acts as a guardian of cellular energy states, coordinating adaptive responses in conditions that deplete ATP, such as hypoxia and ischemia. Silencing PHD2 enhanced the total ATP production rate in both cell lines, with this effect being inhibited by AMPK inhibitors. The ratio of mitochondrial ATP production to glycolytic ATP production indicated enhanced aerobic glycolysis following PHD2 silencing, further supporting the hypothesis that PHD2 regulates energy homeostasis through AMPK.

While PHD2 silencing clearly promotes AMPK phosphorylation, it also affects the expression of other proteins, notably HIF-1α. In normoxic conditions, PHD2 hydroxylates specific residues on HIF-1α, leading to its degradation via the ubiquitin-proteasome pathway. Reduced PHD2 expression stabilizes HIF-1α, enhancing the expression of downstream genes such as VEGF, erythropoietin, GLUT1, and glycolytic enzymes, all of which positively impact wound repair in diabetes. This suggests a potential synergistic effect between AMPK phosphorylation and HIF-1α stabilization, warranting further investigation into their interrelationship.

In conclusion, local silencing of PHD2 in wound tissues activates adaptive hypoxic responses via AMPK phosphorylation, promoting angiogenesis and accelerating wound repair in diabetic conditions. The use of a lentivirus vector, provided by PackGene, was crucial in achieving the efficient delivery of PHD2-shRNA, thereby enabling these findings. These results highlight the potential of targeting PHD2 to enhance wound healing in diabetes through a combination of bioenergetic and hypoxic pathway modulation.

PackGene Biotech is a world-leading CRO and CDMO, excelling in AAV vectors, mRNA, plasmid DNA, and lentiviral vector solutions. Our comprehensive offerings span from vector design and construction to AAV, lentivirus, and mRNA services. With a sharp focus on early-stage drug discovery, preclinical development, and cell and gene therapy trials, we deliver cost-effective, dependable, and scalable production solutions. Leveraging our groundbreaking π-alpha 293 AAV high-yield platform, we amplify AAV production by up to 10-fold, yielding up to 1e+17vg per batch to meet diverse commercial and clinical project needs. Moreover, our tailored mRNA and LNP products and services cater to every stage of drug and vaccine development, from research to GMP production, providing a seamless, end-to-end solution.

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